Lck is a 56-kDa protein tyrosine kinase that is predominantly expressed in T lymphocytes. A member
of the Src kinase family, it has a unique N-terminal region followed by SH3, SH2, and catalytic domains
(1). Lck is an important protein tyrosine kinase in lymphocytes; its overexpression renders T cells
hypersensitive to antigen stimulation (2), and an Lck-deficient T cell line, J.CaM1, exhibits dramatically
reduced protein tyrosine phosphorylation following T cell receptor (TCR) cross-linking (3). Furthermore,
genetic experiments have shown that mice deficient in Lck or expressing a dominant-negative mutant
form of Lck exhibit a severe defect in T cell maturation (4, 5). Lck is localized to the membrane through
myristylation (6) and palmitylation (7-9) and a portion of cellular Lck associates with the cytoplasmic tail
of CD4 via cysteine residues (10-13). CD4 binds to class II major histocompatibility complex molecules on
antigen-presenting cells, and this interaction between CD4 and major histocompatibility complex
activates Lck, perhaps through conformational changes. The Lck associated with CD4 propagates key
biochemical signals in CD4 co-receptor function (14, 15). Like all Src family kinases, Lck is activated and
inhibited by tyrosine phosphorylation; Tyr-394 is the site of stimulatory phosphorylation, whereas
Tyr-505 is the site of inhibitory phosphorylation.
Measurement of Lck Kinase activity
The protocol generally regarded as most sensitive for the quantitative measurement of Lck kinase
activity involves incubation of the Lck kinase sample with substrate (either a natural or synthetic
polypeptide such as poly[Glu, Tyr]4:1), in the presence of Mg2+, Mn2+, and 32P-labeled ATP. The
reaction is terminated by "spotting" a sample onto a filter paper disc, followed by immersion in acid to
precipitate the radiolabeled product. The filter papers are then washed extensively to remove
unincorporated radiolabel and the radioactivity is counted. While sensitive, this method is
labor-intensive, generates hazardous radioactive waste and depends on a radioisotope with a short
half-life. It is particularly unsuitable when kinase assays are only performed on an infrequent basis. The
CycLex® Lck Kinase Assay/Inhibitor Screening Kit uses a horseradish peroxidase-coupled
anti-phosphotyrosine monoclonal antibody as a reporter molecule in a 96-well ELISA format. This assay
provides a non-isotopic, sensitive, and specific method to measure the kinase activity of recombinant
Lck catalytic domain.